The present invention relates to a novel fluorescent dye obtained from a marine animal invertebrate, Holothuria scabra. The present invention also provides a process for the extraction, purification, and characterization of this novel dye, which is a natural dye from marine invertebrate, especially the sea cucumber.
Sea cucumbers are echinoderms, members of the group of spiny skinned animals that also includes Starfishes and sea urchins. The sea cucumber has the following taxonomic position:
Subkingdom: Metazoa
Phylum: Echinodermata
Sub-Phylum: Eleutherozoa
Class: Holothuroidea
Subclass: Aspidochirotacea, Dendrochirotacea, Apodacea
Order: Dendrochirota, Aspidochirota, Elasipoda, Molpadonia and Apoda
Of these orders, the sea cucumber Holothuria scabra belongs to:
Order: Aspidochirota
Family: Holothuroidea
Genus: Holothuria
Species: scabra 
Echinoderms are coelomate invertebrates which are exclusively marine, are never colonial, are unsegmented with a basic pentameric radial, are symmetrical in the adult form, have no head or brain, and are distinguished from all other animals by structural peculiarities of the skeleton and coelom. The class Holothuroidea has animals with a bilaterally symmetrical body and are usually elongated in the oral-aboral axis having a mouth at or near one end and an anus at or near the other end. The body surface is coarse. The endoskeleton is reduced to microscopic spicules or plates embedded in the body wall, the mouth is surrounded by a set of tentacles attached to a water vascular system, the podia or tube feet are usually present and locomotory, the alimentary canal is long and coiled and cloaca usually with respiratory trees and the sexes are usually separate and either gonad single or paired tuft of tubules. They are sedentary and either attach themselves to hard substrate or burrow into soft sediments with anterior and posterior ends projected. They occur in all seas, chiefly in shallow waters. A few species occur in depths greater than 1000 meters. The species Holothuria scabra, also called by some as Metriatyla scabra Jaegea, is widely distributed in East Africa, the Red Sea, the Bay of Bengal, East India, Australia, Japan, the South Pacific, the Philippines, the Indian Ocean, and other Indo-Pacific regions. It is used for human/animal consumption in Sabah, Malaysia and Indonesia and other Indo-Pacific countries.
Pigments are categorized as inorganic or organic. Inorganic pigments are inorganic chemistry compounds which are used for various decorative and painting purposes, etc. Organic pigments such as organic dyes date back to the ancient times. The use of dyes from plants like Brazil wood, long-wood, Persian berry indigo, and madder are reported from near east and far eastern countries even before Biblical times. (George L. Clark, 1966 xe2x80x9cEncyclopaedia of Chemistry, 2nd ed. Pages 833-835). Debra K. Hobson and David S. Wales describe xe2x80x9cGreen dyesxe2x80x9d which are produced as secondary metabolites from some groups of living organisms like fungi, blue green algae, sea urchins, star fishes arthropods, and coral reef coelenterates. (Journal of the Society of Dyers and Colourists (JSDC), 114, 42-44, 1998). These are anthraquinone compounds, historically of crucial importance in the dyestuffs industry. Stainsfilexe2x80x94Dyes A gives a Dye Index of 264 dyes, out of which only six are natural dyes from all types of living organisms. (http://members.pgonline.com/xcx9cbryand/dyes/dyes.htm).
Recently, several patents have issued regarding natural dyes. A majority of them are from plants. Wrolstad, et al. describe a natural colorant from potato extract. (U.S. Pat. No. 6,180,154 issued on Jan. 30, 2001). Shrikhande disclosed the extraction and intensification of anthocyanins from grape pomace and other material. (U.S. Pat. No. 4,452,822 issued Jun. 5, 1984). Lenoble, et al. described a new composition to enhance the red color of anthocyanin pigment. (U.S. Pat. No. 5,908,650 issued Jun. 1, 1999).
Carotenoid-producing bacterial species are disclosed in two U.S. patentsxe2x80x94U.S. Pat. No. 5,935,808 issued on Aug. 10, 1999 to Hirschberg, et al. and U.S. Pat. No. 5,858,761 issued on Jan. 12, 1999 to Tsubokura, et al. Collin disclosed sea cucumber carotenoid processing methods and compositions for lipid fractions. (U.S. Pat. No. 6,055,936 issued on May 2, 2000).
However, these colorants and dyes are not fluorescent. Fluorescent dyes, most of which are synthetic, are disclosed in several U.S. and International patents. These fluorescent dyes have been used in a variety of applications. The amount of patents in this field show the importance of these dyes.
Synthetic parazoanthoxanthin A (m.w. 214.2), emitting fluorescence at lambda (em) 420 nm, was found to be a pure competitive inhibitor of cholinesterases. Sepcic et al., Toxicon, 36(6):937-940, 1998. Welch disclosed a luminescent golf ball. (U.S. Pat. No. 5,989,135 issued on Nov. 23, 1999). White et al. (U.S. Pat. No. 6,110,566 issued on Aug. 29, 2000 and International Patent WO/9920688) described a flexible polyvinyl chloride film that exhibits durable fluorescent color.
Dipietro disclosed the use of fluorescent polymeric pigments in a variety of paints, inks, and textiles. (International Patent WO/9938916). Cramer described a composition containing fluorescent dye for bleaching and brightening polymers. (International Patent EP0206718).
Fluorescent leak detection dye is another utility disclosed by some skilled in the art. (U.S. Pat. No. 6,056,162 issued on May 2, 2000 to Leighley). In addition, Cooper et al. disclosed a full spectrum fluorescent dye composition for the same purpose. (U.S. Pat. No. 6,165,384 issued on Dec. 26, 2000).
Lichtwardt et al. disclose the use of a fluorescent dye in an automated chemical metering system. (U.S. Pat. No. 5,902,749 issued on May 11, 1999).
The reports from marine animals are few. A green fluorescent protein GFP, a novel reporter gene, has been described from the pacific jellyfish Aequova aequora. (Shimomura, et al., Journal of Cellular and Comparative Physiology, 59, 223-239, 1962). GFP is characterized by the presence of a highly fluorescent chromatophore. Purified GFP absorbs blue light maximally at 395 nm with a minor peak at 470 nm and also emits green light. Sepcic et al. reported a fluorescent zoanthid pigment, parazoanthoxanthin A. Toxicon, 36(6):937-940, 1998.
Marine dyes have several uses as dyes on their own and as a part of compositions.
Several authors have disclosed fluorescent dye blends for multiple purposes. (E.g., Burns, et al. in U.S. Pat. No. 5,920,429 issued on Jul. 6, 1999 and Burns, et al. International Patent AU/704112). Marine dye compositions have been used in a number of applications to mark the location of crashed aircraft, life rafts, and military equipment (e.g., rockets). The dye commonly used is fluorescein, which is a water-soluble synthetic dye. Different compositions of the dye for better efficiency and longer duration of fluorescence in diluted form are under trials. (Swinton Robert J., U.S. Pat. No. 5,405,416 issued on Apr. 11, 1995 and International Patent WO/9010044 published on Jul. 7, 1990). Hyosu, et al. has prepared fluorescent colored resin particles. (U.S. Pat. No. 4,016,133 issued on Apr. 5, 1977).
Another use of marine dyes as undersea probes is reported by Crosby, et al. in U.S. Pat. No. 5,321,268 issued on Jun. 14, 1994. The probe device described includes a central optical fiber containing a fluorescent dye enclosed in a transparent or translucent, protective and fouling resistant sheath. This sheath can be attached to a marine animal for collecting data as to light intensity and temperature in regions where the marine animals travel.
Some authors have used UVA in photochemotherapy for skin cancers. Kowalzick, et al. disclose PUVA-bath photochemotherapy in lymphomatoid papulosis (a skin cancer) where UVA treatment has shown improvement. (Elsevier Science B. V., 2000). UVA sunbeds are widely used by patients with psoriasis.
In U.S. Pat. No. 5,210,275 issued on May 11, 1993, Sabatelli disclosed a chromatophore sunscreen composition for preventing sunburns. The chromatophore had the ability to absorb UVA and UVB wavelength radiations.
Fluorescent dyes are very useful in the labeling of molecular probes for fluorescence microscopy. Fluorescence microscopy, also known as reflected light fluorescence or epifluorescence microscopy, is of great value for non-radioactive in situ hybridization because of its high sensitivity and ability to excite three different immunofluorophores with spectrally separated emissions. This makes multiple detections possible. (Chapter II, Nonradioactive In Situ Hybridization Application Manual, Boehringer Mannheim GmbH, Biochemica, printed in Germany, 1992). The principle behind this is that when the specimen is irradiated by the excitation wavelength corresponding to Stoke""s Law, which explains that the wavelength of the fluorescent radiation is always longer than that of the excitation radiation. (Clark, George, xe2x80x9cFluorescencexe2x80x9d, Encyclopaedia of Chemistry, 2nd ed., pages 435-436, 1966; Chapter V, In Situ Hybridization Application Manual, Boehringer Mannheim GmbH, Biochemica, printed in Germany, pages 23-62, 1992; and Olympus Optical Co. Ltd, Tokyo, Japan. Catalogue, xe2x80x9cInstructions BX-FLA Reflected Light Fluorescence Attachmentxe2x80x9d, page 16, 1999 (described variety of non-radioactive fluorochrome stains in use)).
Different stains are used for different excitation cubes of the fluorescent microscope. For example, DAPI (DNA staining, emits blue color), Fluoresceinxe2x80x94dUTP; Hoechest 33258, 33342 are seen under excitation with 330-385 excitation cubes; FITC, Acridine Orange (for DNA, RNA emits greenish/yellowish hues), Auramine under 450-480 excitation cube and Rhodamine, TRITC and Propidium iodide (DNA, emits orange hues) under 510-550 excitation cube.
Rosenblum, et al. used a set of 4,7-dichlororhodamine compounds useful as fluorescent dyes as molecular probes in International Patent WO/0058406 published on Oct. 5, 2000.
LaClair disclosed the synthesis of a fluorescent dye and its application in protein labeling, DNA labeling, single molecule spectroscopy and fluorescence. (U.S. Pat. No. 6,140,041 issued on Oct. 31, 2000 and International Patent WO/9938919).
The present invention adopts a different approach. In particular, the dye disclosed in the present invention is a natural dye and not synthetic. It is from a marine animal and not from a plant or microbes. The partially pure dye is extracted from the cells of the skin of the invertebrate directly. This is a first report for a natural dye from a marine animal which is a fluorescent dye. The marine animal source a holothurian, namely a sea cucumber called Holothuria scabra. Unlike most other fluorescent synthetic dyes known, the dye according to the present invention does not need to be mixed with another dye to obtain different fluorescence hues at different wavelengths. It emits three different colored fluorescence at three different excitation wavelengths, which can have multiple uses. Further, even among the naturally known fluorescent dyes such as the most popular green fluorescent protein (GFP) from a jelly fish, the dye according to the present invention is non-proteinaceous in nature and is more stable at room temperature for months and does not get contaminated by microbes. It also has qualities of a biosurfactant. Another important feature of the dye is that after excitation in the lower UV spectral ranges of wavelengths (UVB), it emits fluorescence in the UVA wavelength range. Both these absorption and emission ranges can be put to selective applications depending upon which UV spectra is preferable in a particular situation.
One important aspect of the dye is its making compositions and kits for non-radioactive labeling of molecular probes and counterstaining. At different wavelength excitations, it gives the effect equivalent to the color of DAPI, FITC and PI. Three in one though it is a single dye. This single dye covers the colors of wavelength spectrum of 123 fluorochromes presently known in the market. (See Bitplane products (Fluorochrome) on the internet at http://www.bitplane.ch/public/support/standard/Fluorochrome.htm).
Yet another aspect of this dye is its use as a fluorochrome stain in epifluorescence microscopy, which is reported here for the first time for any marine natural dye. This application provides a simple and quick method of checking cytogenetical preparations for multiple uses like molecular diagnostics using fluorescent in situ hybridization techniques, rapid diagnosis of biocontamination in tissue cultures, industrial preparations, and water quality check both in laboratory and in wild conditions.
Yet another aspect of the dye is its use as a component of non-radioactive labeling kits for advanced molecular biology applications.
The main object of the present invention is to provide a novel fluorescent dye obtained from the sea cucumber Holothuria scabra. 
Another object of the present invention is to provide a process for the extraction, partial purification, and characterization of the natural dye/pigment form the marine animal Holothuria scabra. 
Yet another object of the present invention is to provide compositions employing the dye obtained from the tissues of Holothuria scabra. 
Yet another object of the present invention is to provide compositions employing the dye obtained from the tissues of Holothuria scabra. 
Yet another object of the present invention is to observe its insecticidal and pesticidal effects.
Yet another object of the present invention is its application for veterinary remedies.
Still another object of the present invention is to provide a dye that emits fluorescence in three different wavelength ranges of the UV and visible light spectra on particular excitation wavelengths.
Another object of the present invention is to obtain and observe the fluorescence and visible spectroscopic analysis and range of emission wavelengths.
Yet another object of the present invention is to observe the three different fluorescence colored emissions of the dye in the UV and visible ranges of epifluorescence microscopy cubes.
Still another object of the present invention is to observe the effect of fluorescence staining of cytogenetical slides to screen chromosomes, cells, and tissues by using the dye of the present invention.
Yet another object of the present invention is biosurfactant nature analysis.
Still another object of the present invention is to develop kits containing the fluorescent dye as a non-radioactive label for molecular probes.
In sum, the present invention provides a novel fluorescent dye obtained from the sea cucumber Holothuria scabra. The invention includes a process for extraction, isolation, and characterization of the dye. Further, the invention provides compositions containing the dye.